LOCUS       sequence-198863-        7870 bp    DNA     circular SYN 15-APR-2025
DEFINITION  human codon optimized TtAgo; pCDNA3 vector backbone, mammalian 
            expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7870)
  AUTHORS   O'Geen H, Ren C, Coggins NB, Bates SL, Segal DJ
  TITLE     Unexpected binding behaviors of bacterial Argonautes in human cells 
            cast doubts on their use as targetable gene regulators.
  JOURNAL   PLoS One. 2018 Mar 27;13(3):e0193818. doi: 
            10.1371/journal.pone.0193818. eCollection 2018.
  PUBMED    29584750
REFERENCE   2  (bases 1 to 7870)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2018-03-27"; volume: "13(3):e0193818. doi"; pages: " 
            10.1371/journal.pone.0193818. eCollection 2018"
COMMENT     Sequence Label: pCDNA-hTtAgo
FEATURES             Location/Qualifiers
     source          1..7870
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        208..411
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     362..382
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     408..432
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             529..549
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             568..633
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2848..2895
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             2914..2970
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(3034..3053)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3078..3126
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3328..3756
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3415..3434)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3625..3646
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3765..3785)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3770..4099
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3950..4085
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4012..4031
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4166..4960
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4220..4239)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4830..4849
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5136..5257
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5173..5192)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5227..5246
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5306..5322)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5306..5322)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5319..5341)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5330..5346
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5354..5360,5361..5378,5379..5384))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5399..5420
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5537..5554)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5708..6296)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5788..5807)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6467..7258,7259..7327))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7090..7109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7328..7432)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7507..7526)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7698..7870,1..207)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
       61 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      121 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      181 tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt
      241 ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt
      301 ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg
      361 acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagagc tcgtttagtg
      421 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
      481 gaccgatcca gcctccggac tctagaggat cgaacccttg ccaccatgcc caagaagaag
      541 aggaaggtgg gaggctccgg aggaagcgac tataaggacc acgacggaga ctacaaggat
      601 catgatattg attacaaaga cgatgacgat aagggtggcg ggtccggcgg tggatccggt
      661 accggaggga gtggtggaag cggcggttct ggtggctcag gcggcagcgg ccggccaatg
      721 aatcacctgg gaaagaccga ggtgttcctg aacagattcg ccctgcggcc cctgaacccc
      781 gaggaactga ggccttggag actggaagtg gtgctggacc ctccacccgg cagagaagag
      841 gtgtaccctc tgctggccca ggtggcaaga cgggctggcg gagtgacagt gcggatggga
      901 gatggactgg ccagctggtc cccacctgaa gtgctggtgc tggaaggcac cctggccaga
      961 atgggccaga cctacgccta ccggctgtac cccaagggca gaaggcccct ggaccctaag
     1021 gatcccggcg agagatctgt gctgagcgcc ctggctagac ggctgctgca ggaaagactg
     1081 cggcgcctgg aaggcgtgtg ggtggaagga ctggccgtgt acagaagaga gcacgccaga
     1141 ggacctggct ggcgggtgct gggcggagct gtgctggatc tgtgggtgtc agatagcggc
     1201 gccttcctgc tggaagtgga ccccgcctac agaatcctgt gcgagatgag cctggaagct
     1261 tggctggctc agggccaccc cctgcctaaa agagtgcgga acgcctacga ccggcggacc
     1321 tgggaactgc tgagactggg cgaagaggac cccaaagagc tgcctctgcc tggcggactg
     1381 agcctgctgg attaccacgc ctctaagggc cggctgcagg gaagagaagg cggcagagtg
     1441 gcttgggtgg ccgatcccaa ggaccccaga aagcccatcc ctcacctgac cggactgctg
     1501 gtgcctgtgc tgaccctgga agatctgcac gaggaagagg gcagcctggc cctgtctctg
     1561 ccctgggagg aaagacgcag acggaccaga gagatcgcca gctggatcgg cagacggctg
     1621 ggactgggaa cacctgaagc tgtgcgggcc caggcctaca gactgagcat ccctaagctg
     1681 atggggcgga gagccgtgtc caaacctgcc gatgctctga gagtgggctt ctacagagcc
     1741 caggaaaccg ctctggccct gctgaggctg gatggcgctc agggatggcc cgagtttctg
     1801 agaagggctc tgctgagagc cttcggcgcc tctggcgctt ctctgagact gcacacactg
     1861 cacgcccacc ctagtcaggg cctggccttt agagaggccc tgcggaaggc caaagaagag
     1921 ggggtgcagg ctgtgctggt gctgacacct cctatggcct gggaagatcg gaaccggctg
     1981 aaagccctgc tgctgaggga aggcctgccc agccagatcc tgaacgtgcc cctgcgggaa
     2041 gaggaacggc acagatggga gaatgccctg ctgggcctgc tggccaaagc tggactgcag
     2101 gtggtggctc tgtctggcgc ctatccagct gagctggccg tgggctttga tgccggcgga
     2161 agagagagct tcagattcgg cggagccgcc tgtgcagtgg gcggagatgg cggacatctg
     2221 ctgtggacac tgcctgaagc ccaggccggc gaaagaatcc cccaggaagt cgtgtgggat
     2281 ctgctggaag agacactgtg ggccttcaga cggaaggccg gcagactgcc tagcagagtg
     2341 ctgctgctgc gcgacggaag agtgccccag gatgaattcg cactggccct ggaagccctg
     2401 gcccgggaag gcattgccta cgatctggtg tccgtgcgga agtccggcgg aggcagagtg
     2461 tatcctgtgc agggcagact ggctgacggc ctgtatgtgc ctctggaaga taagaccttt
     2521 ctgctgctga ccgtgcaccg ggacttcaga ggcaccccca gacctctgaa gctggtgcac
     2581 gaagccggcg acacaccact ggaagctctg gcacaccaga ttttccacct gacccgcctg
     2641 taccccgcca gcggctttgc ctttcctaga ctgcctgccc ctctgcacct ggccgaccgg
     2701 ctcgtgaaag aagtgggcag gctgggcatc cggcacctga aagaggtgga ccgcgagaag
     2761 ctgttcttcg tgggaggctc tggcggatcc gggggatctg gcggaagcgg aggatctgct
     2821 agcggtggag gctccggagg tggatctaaa aggccggcgg ccacgaaaaa ggccggtcag
     2881 gcaaaaaaga aaaagggtgg tagtggaagc ggagctacta acttcagcct gctgaagcag
     2941 gctggagacg tggaggagaa ccctggacct gcggccgcat gaaagggttc gatccctacc
     3001 ggttagtaat gagtttaaac gggggaggct aactgaaaca cggaaggaga caataccgga
     3061 aggaacccgc gctatgacgg caataaaaag acagaataaa acgcacgggt gttgggtcgt
     3121 ttgttcataa acgcggggtt cggtcccagg gctggcactc tgtcgatacc ccaccgagac
     3181 cccattgggg ccaatacgcc cgcgtttctt ccttttcccc accccacccc ccaagttcgg
     3241 gtgaaggccc agggctcgca gccaacgtcg gggcggcagg ccctgccata gcagatctgc
     3301 gcagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg
     3361 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     3421 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     3481 ggggcatccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     3541 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     3601 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     3661 ctatctcggt ctattctttt gatttataag ggattttggg gatttcggcc tattggttaa
     3721 aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt
     3781 agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa
     3841 ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3901 catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct
     3961 aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
     4021 agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg
     4081 aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca
     4141 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4201 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4261 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4321 cctgtccggt gccctgaatg aactgcagga cgaggcagcg cggctatcgt ggctggccac
     4381 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4441 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4501 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4561 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     4621 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     4681 caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     4741 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     4801 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     4861 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     4921 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgcg
     4981 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     5041 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     5101 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     5161 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5221 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     5281 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     5341 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     5401 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     5461 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5521 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5581 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5641 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5701 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5761 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5821 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5881 cgggaagcgt ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg
     5941 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     6001 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     6061 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     6121 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     6181 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     6241 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     6301 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     6361 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     6421 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6481 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6541 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6601 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6661 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6721 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6781 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6841 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6901 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6961 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     7021 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     7081 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     7141 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     7201 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     7261 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     7321 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     7381 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     7441 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtcgac
     7501 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     7561 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     7621 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     7681 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     7741 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     7801 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     7861 agtaacgcca
//
